The present invention relates to the production of paucilamellar lipid vesicles. More particularly, the present invention relates to a method of producing reinforced paucilamellar lipid vesicles with added mechanical strength. These reinforced vesicles have an unstructured central cavity surrounded by 2-10 lipid bilayers. Each of the bilayers has a layer of polyacrylamide lining its innermost side. The central cavity may include a water-immiscible oily phase or an aqueous-soluble material and/or polyacrylamide.
Lipid vesicles are substantially spherical structures made of materials having a high amphiphilic lipid content, e.g., surfactants or phospholipids. There are a variety of uses for lipid vesicles including the use as adjuvants or as carriers for a wide variety of materials. The lipids of these spherical vesicles are organized in lipid bilayers which encapsulate an aqueous volume. The aqueous volume is interspersed between multiple onion-like shells of lipid bilayers (forming multilamellar lipid vesicles or "MLV") or is contained within an amorphous central cavity. The most commonly known lipid vesicles having an amorphous central cavity filled with aqueous medium are the unilamellar lipid vesicles. As the name indicates, unilamellar vesicles have a single lipid bilayer. Large unilamellar vesicles ("LUV") generally have a diameter greater than about 1.mu. while small unilamellar lipid vesicles ("SUV") generally have a diameter of less than 0.2.mu..
Although substantially all the investigation of lipid vesicles in recent years has centered on multilamellar and the two types of unilamellar lipid vesicles, a fourth type of lipid vesicle, the paucilamellar lipid vesicle ("PLV"), exists. This lipid vesicle has barely been studied heretofore and has only been manufactured previously with phospholipids. PLV's consist of about 2 to 10 peripheral bilayers surrounding a large, unstructured central cavity. In all the previously described PLV's, this central cavity was filled with an aqueous solution (Callo and McGrath, Cryobiology 1985, 22(3), pp. 251-267).
Each type of lipid vesicle appears to have certain uses for which it is better adapted. For example, MLV's have a higher structural lipid content then any of the other lipid vesicles so to the extent that a lipid vesicle can encapsulate or carry a lipophilic material within the bilayers, MLV's have been deemed the most advantageous for carrying lipophilic materials. In contrast, the amount of water encapsulated in the aqueous shells between the lipid bilayers of the MLV's is much smaller than the water which can be encapsulated in the central cavity of LUV's, so LUV's have been considered advantageous in transport of aqueous material. However, LUV's, because of their single lipid bilayer structure, are not as physically durable as MLV's and are more subject to enzymatic degradation. SUV's have neither the lipid or aqueous volumes of the MLV's or LUV's but because of their small size have easiest access to cells in tissues.
PLV's, which can be considered a subclass of the MLV's, are a hybrid having features of both MLV's and LUV's. PLV's appear to have advantages as transport vehicles for many uses as compared with the other types of lipid vesicles. In particular, because of the large, unstructured central cavity, PLV's are easily adaptable for transport of large quantities of encapsulated materials. However, the multiple lipid bilayers of the PLV's provides PLV's with the capacity to transport a greater amount of lipophilic material in their bilayers as well as with additional physical strength and resistance to degradation as compared with the single lipid bilayer of the LUV's. However, conventional PLV's are more fragile than MLV's because of the fewer number of bilayers. The central cavity of the PLV's of the present invention can be filled wholly or in part with an apolar oil or wax and then can be used as a vehicle for the transport or storage of lipophilic materials. The amount of lipophilic material which can be transported by the PLV's with an apolar core is much greater than can be transported by MLV's.
Conventional methods of producing multilamellar lipid vesicle start by dissolving the lipids, together with any lipophilic additives, in an organic solvent. The organic solvent is then removed by evaporation using heat or by passing a stream of an inert gas (e.g., nitrogen) over the dissolved lipids. The residue is then hydrated with about an equivalent of an aqueous phase, generally containing electrolytes and additives such as hydrophilic biologically-active materials, to form a separable hydrated lamellar phase. This lamellar phase is then dispersed into an excess of an aqueous phase to form large multilamellar lipid membrane structures. In some variations, different types of particulate matter or structures have been used during the evaporation process to assist in the formation of the lipid residue. Those in the field have shown that by changing the physical structure of the lipid residue, better vesicles form upon hydration. Two recent review publications (Gregoriadis, G., ed. Liposome Technology (CRC, Boca Raton, Fl.), Vols. 1-3 (1984); and Dousset and Douste-Blazy (in Les Liposomes, Puisieux and Delattre, Editors, Techniques et Documentation Lavoisier, Paris, pp.41-73 (1985)) summarize the methods which have been used to make MLV's.
The early lipid vesicle or liposome studies used phospholipids as the lipid source for the bilayers. The reason for this choice was that phospholipids are the principal structural components of natural membranes. However, there are many problems using phospholipids as artificial membranes or vesicles. First, isolated phospholipids are subject to degradation by a large variety of enzymes. Second, the most easily available phospholipids are those from natural sources, e.g., egg yolk lecithin, which contain polyunsaturated acyl chains that are subject to autocatalyzed peroxidation. When peroxidation occurs, the lipid structure breaks down, causing premature release of encapsulated materials and the formation of toxic peroxidation byproducts. This problem can be avoided by hydrogenation but hydrogenation is an expensive process, thereby raising the cost of the starting materials. Cost is a third problem associated with the use of phospholipids on a large scale. For example, a kg of egg yolk lecithin pure enough for pharmacological liposome production presently costs in excess of $1,000.
Recently, there has been some indication that commercially available surfactants might be used to form the lipid bilayer in liposome-like multilamellar lipid vesicles (see, e.g., L'Oreal patent no. 4,217,344, and copending U.S. Pat. application No. 457,571). Both surfactants and phospholipids are amphiphiles, having at least one lipophilic acyl or alkyl group attached to a hydrophilic head group. The head groups are attached to one or more lipophilic chains by ester or ether linkages.
However, even non-phospholipid vesicles may be fragile, resulting in the risk of releasing potentially harmful encapsulated materials to the immediate environment. Therefore, the development of a carrier with added mechanical strength would satisfy a long felt need in the field of material encapsulation and transport.
Accordingly, an object of the invention is to provide paucilamellar lipid vesicles from non-phospholipid materials having added mechanical strength.
Another object of the invention is to provide a method of producing reinforced Paucilamellar lipid vesicles which is rapid, and which involves the use of relatively inexpensive materials
Yet another object of the invention is to provide a stronger vehicle for transport of oil soluble and aqueous materials.
A further object of the invention is to provide a method of encapsulating oily and aqueous materials in lipid vesicles useful for transport of such materials.
These and other objects and features of the invention will be apparent from the following detailed description.